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Abstract

Background

New drugs are constantly sought after to improve the survival of patients with malignant
gliomas. The ideal substance would selectively target tumor cells without eliciting
toxic side effects. Here, we report on the anti-proliferative, anti-migratory, and
anti-invasive properties of the natural, nontoxic compound Curcumin observed in five
human glioblastoma (GBM) cell lines in vitro.

Methods

We used monolayer wound healing assays, modified Boyden chamber trans-well assays,
and cell growth assays to quantify cell migration, invasion, and proliferation in
the absence or presence of Curcumin at various concentrations. Levels of the transcription
factor phospho-STAT3, a potential target of Curcumin, were determined by sandwich-ELISA.
Subsequent effects on transcription of genes regulating the cell cycle were analyzed
by quantitative real-time PCR. Effects on apoptosis were determined by caspase assays.

Results

Curcumin potently inhibited GBM cell proliferation as well as migration and invasion
in all cell lines contingent on dose. Simultaneously, levels of the biologically active
phospho-STAT3 were decreased and correlated with reduced transcription of the cell
cycle regulating gene c-Myc and proliferation marking Ki-67, pointing to a potential
mechanism by which Curcumin slows tumor growth.

Conclusions

Curcumin is part of the diet of millions of people every day and is without known
toxic side effects. Our data show that Curcumin bears anti-proliferative, anti-migratory,
and anti-invasive properties against GBM cells in vitro. These results warrant further in vivo analyses and indicate a potential role of Curcumin in the treatment of malignant gliomas.

Background

Although the introduction of temozolomide treatment in addition to radiotherapy after
surgical resection has improved survival in patients with glioblastoma (GBM), tumor
recurrence is inevitable [1,2]. After tumor recurrence, current as well as novel chemotherapeutic regimens are of
modest benefit, and overall survival rates remain poor [3]. Only a subpopulation of patients (with a methylated O(6)-methylguanine-DNA methyltransferase
(MGMT) gene promoter) may benefit from dose-intensified temozolomide treatment with
added lomustine in terms of overall survival, at the cost of increased toxicity [4]. Therefore, new drugs that are effective in a wider range of GBM patients, most preferably
without inducing additional toxicity, continue to be sought.

Curcumin, derived from the rhizome of the plant Curcuma longa, is the major pharmacologically active component of the spice turmeric and potentially
represents one of those drugs [5]. Being the main ingredient of curries and thus part of the everyday diet of millions
of people, Curcumin is considered a safe agent in humans [5,6]. Recent preclinical as well as first clinical reports have indicated that Curcumin
may be effective in the treatment of various cancers [7-10]. The underlying mechanisms of this efficacy are still under investigation, but recently
an association with the JAK/STAT3 pathway has been proposed [11].

With this study, we aimed to assess the potential effects of treatment with Curcumin
on the hallmarks of GBM, i.e. tumor cell proliferation, migration, and invasion and
to investigate the potential mechanisms of action.

Chemical reagents

Curcumin (94% pure) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) were purchased from LKT (LKT laboratories, St. Paul, MN, USA) and Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), respectively. For stock solutions, Curcumin was dissolved in DMSO at 10 mg/mL and
stored at -20°C; MTT was dissolved in PBS at 5 mg/mL and stored at 4°C.

Cell growth and proliferation assay

Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT)
quantitative colorimetric assay. The viable cell number is directly proportional to
the production of insoluble purple formazan through cleavage of the tetrazolium ring
by mitochondrial enzymes. The coversion can be measured spectrophotometrically (λ
= 560 nm) upon solubilization with 1/24 1 M HCl/95% EtOH.

Cells were seeded at a density of 5,000 cells/well in a 96-well-plate (Greiner Bio-One,
Frickenhausen, Germany) and were allowed to grow in medium containing 10% FCS for
24 hours. Thereafter, cells were incubated with Curcumin at concentrations of 0, 10,
20, and 50 μM. Cells were allowed to grow for various periods of time (6, 12, 24,
48, and 72 hours). Thereafter, cells were incubated with MTT (0.5 mg/ml) for 3 hours.
Cell growth was determined by measuring absorption at indicated periods of time using
a multi-well scanning reader (Tecan GmbH, Crailsheim, Germany). For each experiment,
18 wells were allocated to one treatment or control group.

Wound healing assay

Briefly, 15 - 20 × 105 cells were seeded per well. After the cells were allowed to attach and reach 80% subconfluency,
they were incubated with starvation medium containing 2% FCS for 24 hours prior to
further incubation for 2 hours in starvation medium in the absence (control) or presence
of Curcumin at concentrations of 10, 20, and 50 μM, before a scratch was performed
through the cell monolayer using a yellow pipet tip. Cells were washed with PBS before
photographs of the scratch area were taken in treated and untreated cells using a
Nikon Eclipse TE2000-S microscope (Nikon GmbH, Düsseldorf, Germany). For each well,
two different areas of the scratch were photographed and their location on the dish
was noted. Cells were further incubated for 12 hours in starvation medium before the
exact same areas were re-photographed and cells entering the denuded area were counted.

Briefly, 25,000 cells untreated or treated with Curcumin at concentrations of 10 and
20 μM were seeded into the upper well of the chamber containing serum-free culture
medium. The lower well was filled with culture medium containing 10% FCS. After 24
hours cells on the upper surface of the well were removed and cells on the lower surface
were fixed in 95% ethanol and stained with 0.1% crystal violet. Then, the transmigrated
cells were counted using a Nikon Eclipse TE2000-S microscope (Nikon GmbH, Düsseldorf,
Germany). For each experiment, 10 random high power fields were counted.

Sandwich ELISA

To elucidate the potential mechanism of action, we examined the effect of Curcumin
treatment on the phosphorylation status of the transcription factor STAT3 employing
a sandwich-ELISA kit (PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Antibody Pair #7146; Cell Signaling Technology
Inc., Danvers, MA) according to the manufacturer's advice.

Briefly, after coating the microplate wells, cells were seeded on 10 cm Ø culture
dishes and were incubated for 2 h with Curcumin at 0, 10, 20, or 50 μM, respectively.
Cells were then lysed using ice-cold lysis buffer; the lysates were further sonicated
on ice. Then, 100 μl of the respective lysates were added to a microplate well and
incubated at 37°C for 2 h before the well was washed, and first a detection antibody
(incubation: 1 h) and then a secondary antibody (incubation: 30 min) was added to
each well. After finally adding TMB substrate and STOP solution, absorbance of each
well was measured at λ = 450 nm.

Quantitative real-time PCR

The quantification of mRNA levels was carried out using a real-time fluorescence detection method (TaqMan®) as described previously [13]. Quantitative real-time PCR plots the PCR product on a curve as it accumulates at each cycle of the reaction,
in contrast to conventional PCR, which only displays PCR product at the final cycle.
Total RNA was reversely transcribed using SuperScript™ III reverse transcriptase (Invitrogen GmbH, Karlsruhe, Germany). Subsequently, approximately
30 ng of cDNA were subjected to amplification using an ABI Prism 7500 sequence detection
system with TaqMan® assays (Applied Biosystems Inc, Foster City, CA) according to the manufacturer's advice.
Primers and probes were designed to specifically amplify mRNA of c-Myc (Hs01032443_m1
and Hs00905030_m1), Ki-67 (Hs00153408_m1 and Hs01032435_g1) as well as mRNA of a reference gene, HPRT-1 (Hs99999909_m1).
The ratios of c-Myc or Ki-67 RNA to the reference HPRT-1 represent their relative expression levels. Expression
changes were analyzed with the 2-ΔΔCt method [14].

Caspase cleavage assay

Effector caspase activity of treated and untreated cells was determined as described
previously [15]. Briefly, buffer containing DEVD-7-amino-4-methylcoumarin (AMC) was added to the
lysates of treated (24 h) and untreated cells at a final concentration of 10 μmol/L.
Cells treated with staurosporine (STS) at 3 μM for 16 h served as control. Cells were
incubated for 2 h at 37°C in the dark and the generation of the fluorescent AMC cleavage
product was measured at 380 nm excitation and 465 nm emission, using a fluorescence
plate reader. Fluorescence of blanks containing no cell lysate was subtracted from
the values. Protein content was determined using the Pierce Coomassie Plus Protein
Assay reagent (KMF, Cologne, Germany). Caspase activity is expressed as change in
fluorescence units per microgram protein per hour.

Statistical analysis

All data are expressed as means ± standard error of the mean (SEM) of at least 3 independent
experiments. Statistical differences were evaluated by 1-way ANOVA followed by Tukey's
test using commercially available software (SPSS 17.0; SPSS Inc., Chicago, Ill.).
P values < 0.05 were considered statistically significant.

We hypothesized that the effects on cell proliferation induced by Curcumin may be
explained by its interference with the JAK/STAT3-pathway, as Curcumin was shown to
activate the tyrosine phosphatase SHP-2, a negative regulator of JAK activity [16]. STAT3, activated by JAKs, is a nuclear transcription factor, known to regulate genes
involved in cell cycle progression [17]. We previously reported that STAT3 is constitutively activated in the cell lines
used [18]. In parallel to our observation of reduced cell proliferation, we found reduced transcription
of cell cycle regulating c-Myc already after 2 h of Curcumin treatment (Figure 1C). Correspondingly, quantitative real-time PCR also revealed a decrease of Ki-67 mRNA synthesis after 24 h incubation with Curcumin
(Figure 1D). In concordance with the reduced transcription of cell cycle regulating genes, we
observed a dose-dependent reduction of phosphorylated (active) STAT3 levels after
2 h treatment with Curcumin in all cell lines investigated as determined by ELISA.
When normalized to untreated controls, phospho-STAT3 levels declined to 41-83% after
treatment with 10 μM Curcumin and to 18-35% after treatment with 20 μM Curcumin. Phospho-STAT3
levels eventually diminished to 0-16% after treatment with 50 μM Curcumin (Figure
2A).

Figure 2.Cell migration/invasion and STAT3 levels. A. Bar graphs showing a dose-dependent reduction of intracellular levels of phosphorylated
STAT3 by Curcumin (0, 10 μM, 20 μM, or 50 μM, respectively) determined by ELISA. Data
are from four independent experiments. An asterisk indicates differences that are
statistically significant compared to controls. B. Bar graphs showing restoration of phosphorylated STAT3 levels in MZ-256 cells following
treatment with Curcumin (0, 10, 20, 50 μM) for 2 h as determined by sandwich ELISA.
In cells treated with 10 or 20 μM, normal levels are restored after 12 h, and they
further increase after 24 h. Phosphorylated STAT3 levels remain low for up to 24 h
in cells treated with 50 μM Curcumin. When in contrast cells are treated with Curcumin
continuously for 24 h, phosphorylated STAT3 levels remain low. Data are from three
independent experiments. An asterisk indicates differences that are statistically
significant compared to controls. C. Bar graphs showing a dose-dependent reduction of GBM cell motility by Curcumin (0,
10 μM, or 20 μM, respectively) determined by wound healing assays. Data are from three
independent experiments. An asterisk indicates differences that are statistically
significant compared to controls. D. Bar graphs showing a dose-dependent reduction of the invasive capability of GBM
cells by Curcumin (0, 10 μM, 20 μM, or 50 μM, respectively) determined by modified
Boyden chamber assays. Data are from three independent experiments. An asterisk indicates
differences that are statistically significant compared to controls.

To examine whether STAT3 inhibition by Curcumin is short-lived or long-lasting, we
additionally performed wash out experiments with MZ-256 GBM cells. As indicated in
Figure 2B, the continuous presence of 50 μM Curcumin decreased STAT3 tyrosine-705 phosphorylation
completely for over 24 h, while after withdrawal of the inhibitor the active form
of the transcription factor STAT3 began to resurface at 12 h after the wash out to
reach 60% of its control level after 24 h. This experiment revealed that the estimated
half-life of Curcumin in cultured GBM cells is about 24 h. It can therefore be concluded
that STAT3 inhibition by Curcumin is transient, and Curcumin has to be sustained continuously
for effective treatment.

Curcumin inhibits GBM migration and invasion

Having established a link between Curcumin and phospho-STAT3, we further investigated
the effect of Curcumin on the migratory behavior of GBM cells by performing wound
healing assays. Here, we found that Curcumin treatment significantly inhibited cell
migration in all cell lines in a dose-dependent fashion (Figure 2B). In addition, we performed trans-well assays using modified Boyden chambers to investigate
the effects of Curcumin on the invasive properties of GBM cells. Our findings here
were comparable to the wound healing assays with a dramatically reduced invasiveness
of cells after treatment with Curcumin. At a concentration of 50 μM Curcumin, only
in the MZ-304 cell line there were a few cells invading trough the matrigel membrane;
in all other cell lines, the capability to invade the membrane was completely abolished
(Figure 2C).

Effect of Curcumin on apoptosis in GBM cells

To investigate whether curcumin may not only inhibit cell proliferation, but also
induce apoptosis in GBM cells, a caspase 3-like DEVD cleavage assay was employed with
staurosporine (STS) serving as a positive control for induction of apoptosis. After
treatment with Curcumin, we observed neglibigle induction of effector caspases, whereas
STS induced significant DEVD cleavage activity (Figure 1E).

Discussion

Until today, glioblastomas are incurable malignant tumors. Neither the implementation
of multimodal therapies nor advances in surgical techniques have helped to push median
survival of affected patients above the 2-year boundary [1,19,20]. Therefore, new therapeutic strategies are constantly under investigation. Ideally,
a chemotherapeutic drug would prove efficacious selectively against tumor cells without
inducing unwanted side effects.

Although long-term studies in both animals and humans are lacking, Curcumin, being
a natural compound and the main ingredient of turmeric, commonly known as "curry",
is generally regarded as a safe agent [21]. Therapeutic effects on various cancers have been reported [7,22]. Besides showing an inherent cytotoxicity against malignant cells, Curcumin has additionally
been shown to modulate radio- and chemosensitivity of cancer cells [10,23-25]. With regards to its potential anti-cancer properties, epidemiological data show
a generally low incidence in several types of cancer in populations consuming around
100-200 mg/day [26]. A recent phase I clinical trial in breast cancer demonstrated safety of a daily
intake of 6-8 g Curcumin [27]. Several molecular targets of Curcumin have been implicated in the anticancer effects
of Curcumin, and Curcumin was suggested to affect a number of molecular signaling
cascades [21,28,29].

In this study, we could show that Curcumin potently inhibits proliferation of GBM
cells. Our data further indicate that the efficacy of Curcumin can be explained by
interference with the JAK/STAT3-pathway. STAT3 inhibition represents a novel target
in the treatment of brain tumors. In its active form, STAT3 regulates a number of
pathways important in tumorigenesis including cell cycle progression, migration, and
invasion [30]. In gliomas, there are several reports on a constitutive activation of STAT3 [31]. Normal cells, in contrast to tumor cells are relatively tolerant to interruption
of the STAT3 signaling pathway, making STAT3 an excellent target for molecular therapy
of cancer [32,33]. Gliomas seem to depend on activated STAT3: inhibition of STAT3 is known to suppress
proliferation [34], and STAT3 knockdown reportedly induces apoptosis in glioma cells [35]. Inhibition of STAT3 also leads to reduced transcription of cell cycle regulating
genes such as c-Myc [30]. Here, we demonstrate that Curcumin reduces intracellular levels of biologically
active phosphorylated STAT3 in all GBM cell lines used contingent on dose, which is
paralleled by reduced transcription of c-Myc and Ki-67. Thus, our data indicate that
the effect of Curcumin on GBM proliferation is mediated through interference with
the STAT3 signaling pathway. This conclusion is in line with previous observations
in other cancers [36,37].

We did not observe significant induction of apoptosis in our caspase assays. Therefore,
the robust antiproliferative effects of Curcumin as measured in the MTT assays indeed
reflect an inhibition of cell growth and were not caused by an overall cell loss due
to apoptosis in the cultures. This finding is in line with previous reports demonstrating
cell cycle arrest caused by Curcumin [38].

In addition to cell growth, treatment with Curcumin affected another hallmark of gliomas,
i.e. migration and invasion. We could recently demonstrate that interference with
the JAK/STAT3 pathway inhibits genomic transcription of MMPs and results in decreased
proteolytic activity of MMPs 2 and 9 affecting GBM migration and invasion [18]. Yet, in another report Curcumin inhibited MMP gene expression through interference
with the MAP kinase pathway [39]. It is therefore possible, that the effects of Curcumin could partially be exerted
through several different molecular targets. Due to the variety of potential interactions,
it cannot be ruled out that the observed anti-proliferative effect of Curcumin might
be exerted by interference with another pathway in addition to JAK/STAT3. However,
our study strongly supports the hypothesis that STAT3 is one of the key targets of
Curcumin [36,40]. Likewise, several other groups have reported STAT3 to be associated with migration
and invasion in glial as well as non-glial tumors [41,42]. Finally, STAT3 was most recently considered to be a master regulator of human gliomas
and essential for maintaining tumor initiating capacity and ability to invade the
normal brain [43].

We have shown here that Curcumin potently hampers GBM cell proliferation, migration,
and invasion, and our data suggest that this effect is mediated through interference
with the JAK/STAT3 pathway. Given the fact that STAT3 plays a key role in the mesenchymal
transformation of gliomas, which accompanies aggressive behavior [43], STAT3 may also be a prime target to prevent malignant transformation of low-grade
gliomas. Our data, along with existing reports in the literature, indicate that Curcumin
could become part of the therapeutic armamentarium in the multimodal treatment of
glioma patients. So far, Curcumin represents a safe and low-cost drug, whose application
in clinical practice, even in high doses, in addition to conventional chemotherapeutics
is under investigation in early phase clinical cancer trials [27]. In the future, experimental as well as clinical studies e.g. regarding the combination
of Curcumin and temozolomide or Curcumin and radiation therapy will further elucidate
its therapeutic value in malignant gliomas.

Conclusions

Our data suggest that Curcumin is an effective agent to target GBM cell proliferation
as well as migration and invasion. Its effects are at least partially mediated by
interference with the STAT3 signaling pathway. Exerting anti-tumor properties without
inducing toxicity, Curcumin represents a promising agent against GBM and other cancers.
Further analyses are warranted and necessary to substantiate our findings.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CS conceived the study, performed MTT assays, migration assays, invasion assays, PCR,
and ELISAs, analyzed the data, and drafted the manuscript. MPo performed cell culture
and participated in MTT assays and real-time PCR. MPr performed cell culture and caspase
assays, and participated in migration assays. VS, DK, and JW conceived the study,
supervised the experiments, and helped to draft the manuscript. All authors have read
and approved the manuscript.

Acknowledgements

The authors would like to thank M. Eberhardt for assistance in preparation of the
figures. This work was financially supported by the Deutsche Forschungsgemeinschaft
(German Research Foundation), grant # WE 4358/1-1 to JW and DK.